If you've checked the sequence and the fusion + insert sequence is correct, then cloning it into another vector probably won't help. Is this a C-terminal or N-terminal fusion? If it's N-terminal, could it be that your protein has some kind of N-terminal signal sequence that's cleaved after translation? Maybe you could try a C-terminal fusion instead.

I think you're saying that your backbone-speciifc Ab detected the protein in your wash buffer, but the anti-6x His Ab did not, correct? Did you run samples of the wash buffer on SDS-PAGE and screen by western blot, or just check them by dot blot? If it's a protein conformation problem, perhaps the screen would be negative by dot blot, but be detectable when the protein is in denatured form after SDS-PAGE.

Do you know that your anti-6x His Ab works? We've had some problems with such antibodies -- in some experiments one vendor's anti-6x His MAb doesn't work while another vendor's does...